Functional comparisons of the virus sensor RIG-I from humans, the microbat Myotis daubentonii, and the megabat Rousettus aegyptiacus, and their response to SARS-CoV-2 infection (preprint)

Bats (order Chiroptera) are a major reservoir for emerging and re-emerging zoonotic viruses. Their tolerance towards highly pathogenic human viruses led to the hypothesis that bats may possess an especially active antiviral interferon (IFN) system. Here, we cloned and functionally characterized the virus RNA sensor, Retinoic Acid-Inducible Gene-I (RIG-I), from the "microbat" Myotis daubentonii (suborder Yangochiroptera) and the "megabat" Rousettus aegyptiacus (suborder Yinpterochiroptera), and compared them to the human ortholog. Our data show that the overall sequence and domain organization is highly conserved and that all three RIG-I orthologs can mediate a similar IFN induction in response to viral RNA at 37{degrees} and 39{degrees}C, but not at 30{degrees}C. Like human RIG-I, bat RIG-Is were optimally activated by double stranded RNA containing a 5'-triphosphate end and required Mitochondrial Antiviral-Signalling Protein (MAVS) for antiviral signalling. Moreover, the RIG-I orthologs of humans and of R. aegyptiacus, but not of M. daubentonii, enable innate immune sensing of SARS-CoV-2 infection. Our results thus show that microbats and megabats express a RIG-I that is not substantially different from the human counterpart with respect to function, temperature dependency, antiviral signaling, and RNA ligand properties, and that human and megabat RIG-I are able to sense SARS-CoV-2 infection.

Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome (preprint)

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. A booster vaccination with MVA-MERS-S is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n=1) and the S2 subunit (n=3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. One Sentence Summary A late booster vaccination with the vector vaccine MVA-MERS-S against MERS-CoV is safe and significantly increases humoral immunogenicity including responses to four IgG epitopes.

Serum but not mucosal antibody responses are associated with pre-existing SARS-CoV-2 spike cross-reactive CD4+ T cells following BNT162b2 vaccination in the elderly (preprint)

Advanced age is a main risk factor for severe COVID-19. However, low vaccination efficacy and accelerated waning immunity have been reported in this age group. To elucidate age-related differences in immunogenicity, we analysed human cellular, serological and salivary SARS-CoV-2 spike glycoprotein-specific immune responses to BNT162b2 COVID-19 vaccine in old (69-92 years) and middle-aged (24-57 years) vaccinees compared to natural infection (COVID-19 convalescents, 21-55 years). Serological humoral responses to vaccination exceeded those of convalescents but salivary anti-spike subunit 1 (S1) IgA and neutralizing capacity were less durable in vaccinees. In old vaccinees, we observed that pre-existing spike-specific CD4 + T cells are associated with efficient induction of anti-S1 IgG and neutralizing capacity in serum but not saliva. Our results suggest pre-existing SARS-CoV-2 cross-reactive CD4 + T cells as predictor of an efficient COVID-19 vaccine-induced humoral immune response in old individuals.

Impaired performance of SARS-CoV-2 antigen-detecting rapid tests at elevated temperatures (preprint)

Rapid antigen-detecting tests (Ag-RDTs) can complement molecular diagnostics for COVID-19. The recommended temperature for storage of SARS-CoV-2 Ag-RDTs ranges between 5-30°C. In many countries that would benefit from SARS-CoV-2 Ag-RDTs, mean temperatures exceed 30°C. We assessed analytical sensitivity and specificity of eleven commercially available SARS-CoV-2 Ag-RDTs using different storage and operational temperatures, including (i) long-term storage and testing at recommended conditions, (ii) recommended storage conditions followed by 10 minutes exposure to 37°C and testing at 37°C and (iii) 3 weeks storage followed by testing at 37°C. The limits of detection of SARS-CoV-2 Ag-RDTs under recommended conditions ranged from 8.2×10 5 -7.9×10 7 genome copies/ml of infectious SARS-CoV-2 cell culture supernatant. Despite long-term storage at recommended conditions, 10 minutes pre-incubation of Ag-RDTs and testing at 37°C resulted in about ten-fold reduced sensitivity for 46% of SARS-CoV-2 Ag-RDTs, including both Ag-RDTs currently listed for emergency use by the World Health Organization. After 3 weeks of storage at 37°C, 73% of SARS-CoV-2 Ag-RDTs exhibited about ten-fold reduced sensitivity. Specificity of SARS-CoV-2 Ag-RDTs using cell culture-derived human coronaviruses HCoV-229E and HCoV-OC43 was not affected by storage and testing at 37°C. In summary, short- and long-term exposure to elevated temperatures likely impairs sensitivity of several SARS-CoV-2 Ag-RDTs that may translate to false-negative test results at clinically relevant virus concentrations compatible with inter-individual transmission. Ensuring appropriate transport and storage conditions, and development of tests that are more robust across temperature fluctuations will be important for accurate use of SARS-CoV-2 Ag-RDTs in tropical settings.

Functional comparison of MERS-coronavirus lineages reveals increased zoonotic potential of the recombinant lineage 5 (preprint)

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pneumonia in humans. The virus is enzootic in dromedary camels across the Middle East and Africa. It is acquired through animal contact and undergoes limited onward transmission particularly in hospitals. Because of this initial potential for human-to-human transmission, we monitor the virus for phenotypic changes related to its pandemic potential. Potential phenotypic changes have been suspected since the year 2015, when a novel recombinant clade (MERS-CoV lineage 5) caused large nosocomial outbreaks in Saudi Arabia and South Korea that effectively swept other, hitherto co-circulating viral lineages. To this day, lineage 5 remains the only circulating MERS-CoV lineage on the Arabian Peninsula. In spite of available sequence data, no studies of viral phenotype have been carried out to date. Here we performed a comprehensive in-vitro and ex-vivo comparison of live virus isolates taken in Saudi Arabia immediately before and after the shift toward lineage 5. We characterized seven isolates representing the recombination-parental lineage 3, eight isolates representing parental lineage 4, as well as eight isolates representing lineage 5. Replication of lineage 5 viruses is significantly increased over isolates from parental lineages in cell culture and ex-vivo lung models. Transcriptional profiling by real-time RT-PCR shows that several key immune genes (IFNb1, CCL5, IFNL1) are significantly less induced in lung cells infected with lineage 5 MERS-CoV compared to parental strains. In IFN receptor knock out cells, as well as under chemical inhibition of IFN signalling, the differences in replication level between lineage 5 and parental lineages are reduced, suggesting that phenotypic differences may be determined by IFN antagonism. Concordantly, lineage 5 shows increased resilience against interferon (IFN) pre-treatment of Calu-3 cells and maintains a 10-fold higher replication level under low and high concentrations of IFN. Reduced immune activation combined with enhanced virus replication and IFN resilience may explain the dominance of lineage 5 on the Arabian Peninsula. This phenotypic difference is highly relevant with regard to pandemic potential, and has remained undiscovered in spite of viral sequence surveillance.